【摘要】 目的:观察微球培养的人脂肪间充质干细胞在特定诱导条件下向软骨细胞的分化情况,探讨其作为组织工程化软骨种子细胞的可行性。方法:实验于2006-03/08在湘雅医院中心实验室完成。①实验材料:皮下脂肪组织来源于股骨颈骨折手术患者6例,对本实验均知情同意。②实验方法:取皮下脂肪,分离培养脂肪间充质干细胞,胰酶消化,取传至第6代的细胞,密度调整为1×1010L-1进行接种培养,细胞贴附后加入软骨细胞诱导剂(含10μg/L转化生长因子β1、37.5mg/L维生素C、6.25mg/L胰岛素、6.25mg/L转铁蛋白、10-7mol/L地塞米松、1%新生牛血清的高糖DMEM),倒置显微镜下观察细胞团的变化。③实验评估:诱导14d后,将细胞吹散,细胞爬片,免疫组化染色检测Ⅱ型胶原的表达,甲苯胺兰染色观察细胞内异染情况,RT-PCR检测Ⅱ型胶原蛋白和软骨蛋白聚糖aggrecan mRNA的表达。结果:①脂肪间充质干细胞向软骨细胞诱导后的形态:诱导3d后,细胞团向内呈放射状聚集。②诱导后Ⅱ型胶原的表达及细胞内异染情况:诱导14d后,Ⅱ型胶原在胞浆、胞膜及细胞外基质中均有表达,呈棕黄色;甲苯胺兰染色显示胞浆内呈蓝色异染。③诱导前后Ⅱ型胶原蛋白和aggrecan mRNA的表达:诱导前脂肪间充质干细胞无Ⅱ型胶原蛋白和aggrecan mRNA表达,而诱导14d后二者均有较高表达。结论:在微球状态下可成功诱导脂肪间充质干细胞向软骨细胞分化。更多还原

【Abstract】 AIM:To investigate the chondrogenic differentiation from human adipose tissue-derived mesenchymal stem cells(ADMSCs)using the micromass culture technique and the feasibility of ADMSCs as seeding cells in cartilage tissue engineering.METHODS:The experiment was performed at the Central Laboratory of Xiangya Hospital from March to August 2006.①Subcutaneous tissue was collected from 6 patients with femoral neck fracture.All patients knew the experiment.②Adipose was attained from subcutaneous tissue.The ADMSCs were isolated and cultured by trypsin digestion method.The six generation ADMSCs were cultured at the density of 1×1010 L-1,and then after adherence these cells were treated with chondrogenic media containing 10 μg/L transforming growth factor(TGF)β1,37.5 mg/L vitamin C,6.25 mg/L insulin,6.25 mg/L transferring,10-7 mol/L dexamethasone,high-glucose DMEM with 1% new-born calf serum(NBCS).Cell mass was observed with inverted microscope.③14 days later,the induced cells were blown to spread out and adhered to the plate bottom.Collagen type Ⅱ expression and metachromatic condition were determined by immunohistochemical method and toluidine blue stain.Expression of collagen type Ⅱ and proteoglycan aggrecan mRNA were measured by RT-PCR.RESULTS:①ADMSCs formation after chondrogenic differentiation:3 days after induction,ADMSCs were radial clump inwardly.②Collagen type Ⅱ expression and metachromatic condition:After induced for 14 days,collagen type Ⅱ expressed in kytoplasm,cell membrane and extracellular matrix,displaying dark yellow.Blue chondrogenic ADMSCs were seen by toluidine blue stain.③Expression of collagen type Ⅱ and proteoglycan aggrecan mRNA before and after induction:Expression of collagen type Ⅱ and proteoglycan aggrecan mRNA were not found in ADMSCs before induction,but highly expressed 14 days later.CONCLUSION:Under micromass culture condition,the ADMSCs can successfully differentiate into chondrocytes.更多还原